Once the blood coagulation process has been initiated, the coagulation cascade goes through the stages of sequentially activating various proenzymes (zymogens) in the blood into their active forms, the serine proteases. This includes, among others, factor XII/XIIa, factor XII/XIa, factor IX/IXa, factor XIXa, factor VII/VIIa, and prothrombin/thrombin. Most of these enzymes are active in the physiological state only if they are associated in a complex on a membrane surface. Ca ions are involved in many of these processes. Blood coagulation follows either the intrinsic pathway, in which case all protein components are present in the blood, or the extrinsic pathway, in which the cell membrane tissue factor plays a critical role. Closure of the wound finally takes place as a result of the conversion of fibrinogen into fibrin through the action of thrombin.
The prothrombinase complex is responsible for activating prothrombin to form thrombin. Thrombin is an important enzyme which can act both as a procoagulant and as an anticoagulant. The prothrombinase complex, in which, among others, factor Va (as a cofactor) and factor Xa (as serine protease) participate, assembles in a Ca-dependent association on the surface of phospholipids. It is hypothesized that the catalytic component of the prothrombinase complex is factor Xa.
Factor X (also called Stuart-Prower factor or Prower factor) is a vitamin K-dependent coagulation glycoprotein which participates in the intrinsic and extrinsic blood coagulation cascade. The primary translation product of factor X (pre-pro-FX) contains 488 amino acids and is synthesized by the liver of by human hepatoma cells first as a single-chain 75 kD precursor protein. In the plasma, factor X is present mainly as a double-chain molecule (Fair et al., Blood 64 (1984), pp. 194-204).
During the biosynthesis, after cleavage of the presequence by a signal peptidase (between Ser23 and Leu24) and the propeptide (between Arg40 and Ala41), the single-chain factor X molecule is cleaved by processing and deletion of the tripeptide Argl 80-Lysl 81-Arg 182 into the double-chain form which comprises an approximately 22 kD light chain and an approximately 50 kD heavy chain, the two chains being connected by way of a disulfide bridge (FIG. 1). Factor X therefore circulates in the plasma as a double-chain molecule.
During the blood coagulation process, factor X is converted from the inactive zymogen into active protease factor Xa through limited proteolytic action, in the course of which the activation of factor X to form Factor Xa can take place in one of 2 membrane-bound complexes: the extrinsic factor VIIa/tissue factor complex or the intrinsic factor VIIIa/factor IXa phospholipid Ca complex, the so-called “tenase complex” (Mertens et al., Biochem. J. 185 (1980), pp. 647-658). A proteolytic cleavage between amino acids Arg234 and Ile235 leads to the release of a 52 amino acids long activation peptide from the N-terminus of the heavy chain and thus to the formation of the active enzyme, factor Xa. The catalytic center of factor Xa is located on the heavy chain.
The activation via the factor VIIa-TF (extrinsic) complex leads to the formation of factor Xaα (35 kD) and factor Xaβ (31 kD), and, if the concentrations of factor VIIa in the complex are low, a polypeptide of 42 kD is present as well.
The formation of factor Xaα takes place via a cleavage at Arg234/Ile 235 of the heavy chain and represents the activation of factor X to form factor Xa. The presence of factor Xaβ presumably results from an autocatalytic cleavage at Arg469/Gly470 in the C-terminus of the heavy chain of factor Xaα and the cleavage of a 4.5 kD peptide. Like factor Xaα, factor Xaβ also has catalytic activity. It was shown, however, that during the cleavage of factor Xaα to form Xaβ, a plasminogen receptor binding site forms and that factor Xaβ may also have fibrinolytic activity and may participate as a cofactor in the fibrinolysis. The conversion of factor Xaα into factor Xaβ, however, proceeds more slowly than the formation of thrombin, as a result of which the initiation of the fibrinolysis prior to the formation of a blood clot is prevented (Pryzdial et al., J. Biol. Chen 271 (1996), pp. 16614-16620; Pryzdial et al., J. Biol. Chem. 271 (1996), pp. 16621-16626).
The 42 kD polypeptide results from a processing in the C terminus of the heavy chain between Arg426 and Gly470 without prior processing between Arg234 and lie 235. Like a factor Xaγ fragment, this intermediate which forms as a result of proteolysis at Lys370 also does not have any catalytic activity (Mertens et al., Biochem. J. 185 (1980), pp. 647-658; Pryzdial et al., J. Biol. Chem. 271 (1996), pp. 16614-16620).
The activation of factor X in the intrinsic pathway is catalyzed by the factor IXa-fctor VIIIa complex. During the activation, the same processing products are obtained, but the factor Xaβ product is obtained in a greater yield than other factor X processing products (Jesty et al., J. Biol. Chem. 249 (1974), p. 5614).
In vitro, factor X can be activated, for example, by means of Russell's Viper Venom (RVV) or trypsin (Bajaj et al., J. Biol. Chem. 248, (1973), pp. 7729-7741) or purified physiological activators, such as FVIIa/TF complex or factor IXa/factor VIIIa complex (Mertens et al., Biochem. J. 185 (1980), pp. 647-658).
In most cases, commercially available factor X products from plasma contain a mixture of factor Xaα and factor Xaβ since after the activation of factor X to form factor Xa, primarily factor Xaα forms, which, in turn, is cleaved in an autocatalytic process to form factor Xaβ.
To produce a uniform factor Xa product with a high molecular integrity, EP 0 651 054 proposed that factor X be activated over a relatively long period of time with RVV, with the result that the resulting final product contained mainly factor Xaβ. Both the by-products, for example, factor Xaα, and the protease were subsequently removed in several chromatographic steps.
The cDNA for factor X was isolated and characterized (Leytus et al., Proc. Natl. Acad. Sci. USA 82 (1984), pp. 3699-3702; Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985), pp. 3591-3595). Human factor X was expressed in vitro in various cell types, such as human embryonal kidney cells or CHO cells (Wolf et al., J. Biol. Chem. 266 (1991), pp. 13726-13730). It was found, however, that in the recombinant expression of human factor X, in contrast to the in vivo situation, the processing in position Arg40/Ala41 is inefficient and that different N termini form on the light chain of factor X (Wolf et al., J. Biol. Chem. 266 (1991), pp. 13726-13730). In vitro, recombinant factor X (rFX) was activated by means of RVV to form recombinant factor Xa (rFXa) or rFXa was directly expressed, in the course of which the activation peptide of amino acid 183 to amino acid 234 was deleted and replaced with a tripeptide to enable processing directly into a double-chain rFXa form. Approximately 70% of the purified rFX were processed to form a light and a heavy chain, and the remaining 30% constituted single-chain rFX with 75 kD. Although the direct expression or rFXa did lead to the formation of active factor Xa, it also led to inactive intermediates. In addition, Wolf et al. (J. Biol. Chem. 266 (1991), pp. 13726-13730) also observed a decreased activity of recombinant factor X, which they attributed to the inferior activation ability of rFX through RVV and to the inactive population of proteins and polypeptides of the single-chain precursor molecule. In particular, they found that rFXa, when expressed by recombinant cells, is highly unstable, which they attributed to the high autoproteolytic rate.
WO 98/38317 describes factor X analogs, in which the amino acids can be modified between Glu228 and Arg234, as a result of which these constructs can be activated, for example, by proteases, such as furin.
To study the function of the C-terminal peptide of factor Xaα, Eby et al. (Blood 80 (Suppl. 1) (1992), pp. 1214 A) introduced a stop codon in position Gly430 of the factor X sequence. They did not, however, find a difference between the activation rate of factor Xa (FXaα) with a β-peptide and a deletion mutant without a β-peptide (FXaB).
Factor Xa is an important component of the prothrombinase complex and is therefore used for the quick arrest of bleeding as well as in patients with blood coagulation disorders, such as hemophilia. Especially in the treatment of patients suffering from hemophilia, which is characterized by a factor VIII or a factor IX deficiency, with factor concentrates that are produced from plasma, a complication that frequently arises is that inhibitory antibodies to these factors are formed. Therefore, a number of alternatives were developed to treat patients suffering from hemophilia with factors with a bypass activity. Thus, for example, the use of prothrombin complex concentrate, partially activated protirombinase complex (APPC), factor VIIa, or FEIBA has been proposed. Commercial preparations with factor VIII inhibitory bypass activity include, for example, FEIBA® or Autoplex®. FEIBA, for example, contains comparable units of factor II, factor VII, factor IX, factor X, and FEIBA, small quantities of factor VIII and factor V, and traces of activated coagulation factors, such as thrombin and factor Xa and/or a factor with factor Xa-like activity (Elsinger, Activated Prothrombin Complex Concentrates. Eds. Marian Russo, Mandelli (1982), pp. 77-87). Elsinger especially stresses the importance of a “factor Xa-like” activity in FEIBA. The factor VIII inhibitory bypass activity was demonstrated by Giles et al. (British J. Hematology 9 (1988), pp. 491-497) in the animal model in particular for a combination of purified factor Xa and phospholipids.
Thus, there is a considerable need and a number of different fields of application for factor X/Xa or factor X/Xa-like proteins, either by themselves or as a component of a coagulation complex in hemostatic therapy. Compared to the half-life of zymogen, the half-life of factor Xa is considerably reduced both in vivo and in vitro. Thus, for example, factor X can be stably stored in glycerol for 18 months while under the same conditions, factor Xa is stable only for 5 months (Bajaj et al., J. Biol. Chem. 248 (1973), pp. 7729-2241), or, if stored in glycerol at 4° C. for 8 months, it shows a reduction of the activity by more than 60% (Teng et al., Thrombosis Res. 22 (1981), pp. 213-220). In serum, the half-life of factor Xa is only 30 seconds.
Due to the instability of factor Xa, it has been proposed that factor X preparations be administered (U.S. Pat. No. 4,501,731). In cases of life-threatening bleeding, in particular in patients suffering from hemophilia, however, an administration of factor X has no effect since, due to the lack of the functional “tenase complex,” it is not possible for an effective activation of factor X into factor Xa to take place in the intrinsic blood coagulation pathway and since an activation by way of the extrinsic pathway often takes place too slowly to have a rapid effect. Furthermore, patients suffering from hemophilia have a sufficient supply of factor X; however, compared to factor Xa, factor X has a prothrombinase activity that is 1000 times lower. In cases of this type, activated factor Xa must be administered directly, possibly in combination with phospholipids, such as described by Giles et al. (British J.
Haematology 9 (1988). pp. 491-497), or with other coagulation factors, for example, with factor VIII inhibitory bypass activity.
In the production of factor Xa from factor X, the activation has so far been triggered mainly by means of nonphysiological activators of animal origin, such as RVV or trypsin, but this means that care has to be taken to ensure with absolute certainty that the final product is completely free from these proteases. As already mentioned above, during the activation of factor X to factor Xa, a large number of inactive intermediates is formed (Bajaj et al., J. Biol. Chem. 248 (1973), pp. 7729-7741, Mertens et al., Biochem. J. 185 (1980), pp. 647-658). The presence of such intermediates leads to a decrease of the specific activity of the product and potentially even to the type of intermediates that might serve as antagonists of the active serine protease. Thus, to produce a uniform, pure product with a high specific activity by means of conventional methods, time-consuming and complicated procedures for the activation and chromatographic purification are required.